The present invention relates to a rhodamine derivative useful as a reagent for measurement of nitric oxide, and a reagent for nitric oxide measurement that comprises said compound.
Nitric oxide (NO) is an unstable radical species of a short life, and has been elucidated to have important functions as a physiological active substance in a living body (Chemistry Today [Gendai Kagakul], April, 1994, Special Edition; Pharmacia, May, 1997, Special Edition). Methods for measuring nitric oxide are roughly classified into indirect methods, which measure NO2xe2x88x92 and NO3xe2x88x92 as oxidative degradation products of nitric oxide, and methods based on direct measurement of nitric oxide. The direct methods have been desired from viewpoints of detection and quantification of nitric oxide under physiological conditions. However, any specific and highly sensitive detection method that can be applied to in vitro systems has not been developed so far.
As typical methods, there are known, for example, the chemiluminescence method utilizing the luminescence generated by ozone oxidation of NO radicals (Palmer R. M., et al., Nature, 327, pp.524-526, 1987), a method determining absorption spectrum of metHb which is produced by oxidation of oxyhemoglobin (O2Hb) (Kelm M., et al., Circ. Res. 66, pp.1561-1575, 1990), a method for quantification utilizing the flow of electric current produced in oxidation when electrodes are placed in a tissue (Shibuki K., Neurosci. Res. 9, pp.69-76, 1990; Malinski, and T., Nature, 358, pp.676-678, 1982), the Griess reaction method (Green L. C., et al., Anal. Biochem., 126, pp.131-138, 1992) and so forth (as reviews, see, xe2x80x9cApproaches From The Newest Medicine [Saishin Igaku Kara No Approach] 12, NOxe2x80x9d, Ed. by Noboru Toda, pp.42-52, Section 3, Tetsuo Nagano, Measuring Method of NO, published by Medical View Co., Ltd; Archer, S., FASEB J., 7, pp.349-360,1993).
The Griess reaction method achieves the detection by using azo coupling of a diazonium salt compound and naphthylethylenediamine in the presence of NO2xe2x88x92 that is produced by oxidation of a nitric oxide radical. Although this method does not achieve direct measurement of nitric oxide radicals, the method is advantageous since it requires no special apparatuses or techniques. Moreover, this method also has a characteristic feature that nitric oxide-related metabolites can be quantified, since NO3xe2x88x92 can be measured after being reduced to NO2xe2x88x92 with cadmium (Stainton M. P., Anal. Chem., 46, p.1616, 1974; Green L. C., et al., Anal. Biochem., 126, pp.131-138, 1982) or hydrazine (Sawicki, C. R. and Scaringelli, F. P., Microchem. J., 16, pp.657-672, 1971).
2,3-Diaminonaphthalene is known as a reagent for measuring nitric oxide by detecting NO2xe2x88x92 in a similar manner to the Griess reaction method. This reagent reacts with NO2xe2x88x92 under an acidic condition to form a fluorescent adduct, naphthalenetriazole (chemical name: 1-[H]-naphtho[2,3-d]triazole, Wiersma J. H., Anal. Lett., 3, pp.123-132, 1970). The conditions for the reaction of 2,3-diaminonaphthalene with NO2xe2x88x92 have been studied in detail. The reaction proceeds most quickly at pH 2 or lower and completes in approximately 5 minutes at room temperature (Wiersma J. H., Anal. Lett., 3, pp. 123-132, 1970; Sawicki, C. R., Anal. Lett., 4, pp.761-775, 1971). The generated adduct emits fluorescence most efficiently at pH 10 or higher (Damiani, P. and Burini, G., Talanta, 8, pp.649-652, 1986).
The method for measuring nitric oxide using the 2,3-diaminonaphthalene is characterized in that a detection limit is about several tens nanomoles and sensitivity is 50 to 100 times higher than that of the Griess reaction method (Misko, T. P., Anal. Biochem. 214, pp.11-16, 1993). This method is also excellent since it can be carried out conveniently without need of any special apparatuses or techniques (as a review of the above methods, see, DOJIN News, No. 74, Information Measurement Reagents for NO: 2,3-Diaminonaphthalene, published by Dojindo Laboratories Co., Ltd., 1995). However, this method does not utilizes nitric oxide, per se, but its oxidation product NO2xe2x88x92 as the reaction species, and accordingly, the method is rather indirect as compared to the direct methods for measuring nitric oxide. In addition, since the reaction of 2,3-diaminonaphthalene and NO2xe2x88x92 is performed under a strongly acidic condition (pH 2 or lower), it has a problem that the method cannot be used for detection and quantification of nitric oxide under a physiological condition.
The inventors of the present invention conducted researches to provide means for direct measurement of nitric oxide with high sensitivity under a physiological condition. As a result, the inventors found that 2,3-diaminonaphthalene or a derivative thereof efficiently reacts with nitric oxide to give fluorescent naphthalenetriazole or its derivative, even under a neutral condition, in the presence of an oxygen source such as dissolved oxygen or oxide compounds (for example, PTIO and its derivatives such as carboxy-PTIO). Moreover, the inventors also found that a method for measuring nitric oxide employing this reaction gave extremely high detection sensitivity and achieved accurate quantification of a trace amount of nitric oxide (see, the specification of Japanese Patent Unexamined Publication (Kokai) No. 9-043153/1997).
However, the aforementioned method utilizing 2,3-diaminonaphthalene needs irradiation by excitation light of a short wavelength such as about 370 to 390 nm for the detection of fluorescence, and accordingly, cells and tissues in a measurement system may possibly be damaged. The method also has a problem in that strong autofluorescence of cells may affect the measurement. Moreover, the fluorescent triazole compound produced from 2,3-diaminonaphthalene does not necessarily have sufficient fluorescence intensity, and for this reason, it is difficult to accurately measure fluorescence in individual cells by using conventional fluorescence microscopes. There is also a problem in that 2,3-diaminonaphthalene itself has a relatively simple chemical structure and is not suitable as a fundamental structure for various chemical modification so as to be localized inside of cells.
The inventors of the present invention proposed two methods for measurement of nitric oxide that successively solve these problems.
One of the methods utilizes a diaminofluorescein derivative (hereafter also referred to as xe2x80x9cDAFxe2x80x9d in the specification, Japanese Patent Unexamined Publication (Kokai) No. 10-226688/1998). This method utilizing DAF is much excellent in reactivity with nitric oxide and measurement sensitivity. The method enables measurement of nitric oxide with excitation light of a long wavelength that does not damage living tissues and cells, and accurate measurement of nitric oxide existing in inside of cells for each individual cell, which are characteristic features of the aforementioned method. However, since a part of fluorescence wavelength range of the triazole derivatives (hereafter also referred to as xe2x80x9cDAF-Txe2x80x9d) that are produced by the reaction of DAF with nitric oxide overlaps with the autofluorescence range of cells, the method may sometimes fail to accurately measure nitric oxide in certain types of samples. Further, since the fluorescence of DAF-T may be attenuated from weakly acidic to acidic region, a problem also arises in that accurate measurement over a wide pH range cannot be conducted.
The second method utilizes a diaminorhodamine derivative (hereafter also referred to as xe2x80x9cDARxe2x80x9d, International Patent Publication WO99/01447). This method is also based on the measurement of fluorescence of a triazole derivative (hereafter also referred to as xe2x80x9cDAR-Txe2x80x9d) which is produced by the reaction of DAR with nitric oxide. The peak of the fluorescence spectrum of DAR-T lies around 580 nm (excitation wavelength: 565 nm), while the peak of the fluorescence spectrum of the aforementioned DAF-T is observed around 515 nm (excitation wavelength: 495 nm). Accordingly, measurements without being influenced by autofluorescence of cells can be performed by using DAR. Furthermore, since DAR-T can maintain a certain level or higher intensity of fluorescence in an acidic region as well as in a basic or a neutral region, it enables measurement of nitric oxide over a wide pH range. However, in the method utilizing DAR, fluorescence intensity of some DAR-T may sometimes slightly fluctuate depending on pH, and a problem arises that accurate measurement cannot be performed when a sample is measured whose pH is possibly fluctuate during measurement, e.g., a tissue of a patient with an ischemic disease. There is also a problem that DAR-T has lower fluorescence intensity as compared to the class of DAF-T.
An object of the present invention is to provide a compound useful for the measurement of nitric oxide. More specifically, the object is to provide a compound which has higher fluorescence intensity and whose fluorescent intensity is not fluctuated by pH, which compound is based on the aforementioned DAR that can maintain a certain level of fluorescence intensity in an acidic region as well as in a basic and a neutral region without being influenced by the autofluorescence. Another object of the present invention is to provide a reagent for measurement of nitric oxide comprising a compound having the aforementioned characteristics.
The inventors of the present invention conducted various researches to achieve the foregoing objects. As a result, they found that the rhodamine derivatives of the following structures readily reacted with nitric oxide in a sample for measurement to give triazole derivatives having high fluorescence intensity. They also found that the triazole derivatives had a substantially constant level of fluorescence intensity over a wide pH range from basic to acidic condition, and that their fluorescence intensities were improved. The present invention was achieved on the basis of these findings.
The present invention thus provides compounds represented by the following formula (I) or (II): 
wherein, in the formula (I), R1, R2, R3, and R4 independently represent methyl group or ethyl group; and in the formula (II), R5, R6, R7, and R8 independently represent methyl group or ethyl group and Xxe2x88x92 represents an anion. According to preferred embodiments of the present invention, there are provided: the compounds of the formula (I) wherein R1, R2, R3, and R4 are ethyl groups; the compounds of the formula (I) wherein R1, R2, R3, and R4 are methyl groups; the compounds of the formula (II) wherein R5, R6, R7, and R8 are methyl groups; and the compounds of the formula (II) wherein R5, R6, R7 and R8 are methyl groups and Xxe2x88x92 is Ixe2x88x92. According to another aspect of the present invention, there are provided reagents for measurement of nitric oxide which comprises a compound represented by the aforementioned formula (I) or formula (II).
According to a further aspect of the present invention, there are provided compounds represented by the following formula (III) or (IV): 
wherein, in the formula (III), R11, R12, R13, and R14 independently represent methyl group or ethyl group; and in the formula (IV), R15, R16, R17, and R18 independently represent methyl group or ethyl group and Yxe2x88x92 represents an anion. According to preferred embodiments of the aforementioned invention, there are provided: the compounds wherein R11, R12, R13, and R14 are ethyl groups; the compounds wherein R11, R12, R13 and R14 are methyl groups; the aforementioned compounds wherein R15, R16, R17 and R18 are methyl groups; and the aforementioned compounds wherein R15, R16, R17 and R18 are methyl groups and Yxe2x88x92 is Ixe2x88x92.
According to a still further aspect of the present invention, there are provided methods for measurement of nitric oxide, which comprise step (1) in which a compound represented by the aforementioned formula (I) or (II) is reacted with nitric oxide, and step (2) in which a compound represented by the formula (III) or (IV) produced in the step (1) is detected.